Peripheral arterial disease (PAD), caused by atherosclerosis that impairs blood flow to the lower extremities, is a major health problem. Currently there are no pharmacological therapies for PAD that have the ability to increase perfusion and correct the impaired blood flow. To date, dozens of trials of therapeutic angiogenesis in humans covering thousands of patients have almost uniformly failed. We pose that human angiogenesis treatment regimens for PAD were deployed without an adequate appreciation of the complexities that regulate the numerous competing processes at the molecular, cellular, tissue/organ, and whole body levels; thus accounting for clinical trial failurs. To understand these phenomena at the fundamental level and to develop novel therapeutic approaches, quantitative computational systems biology approaches synergistically combined with experimental measurements are not only desirable but absolutely necessary. The broad goal of the project is to gain a quantitative understanding of angiogenesis in PAD, using a highly synergistic combination of predictive computational modeling and in vivo experiments; and further, using this knowledge, to design improved and novel human therapeutics. This project presents a paradigm shift from stimulating angiogenesis purely by administration of pro-angiogenic molecules to stimulating angiogenesis by inhibiting endogenous anti-angiogenic molecules. In Specific Aim 1 we will expand systems characterization of non-ischemic and ischemic muscle to understand the impact of previously unaccounted-for endogenous anti-angiogenic isoforms of vascular endothelial growth factor VEGFxxxb. We will formulate computational models to predict the dynamics of VEGF receptor-ligand activation in healthy non-ischemic mouse muscle and following hind-limb ischemia (HLI) and in human patients to explore novel potential therapeutic targets for PAD. In Specific Aim 2 we will characterize endogenous anti-angiogenic protein thrombospondin-1 (TSP1) as a potential therapeutic target for PAD. TSP1 is a strong anti-angiogenic agent transducing signals through multiple receptors; TSP1 has been shown to be elevated in PAD. We will develop, de novo, computational models of signal transduction through TSP1 receptors, also considering their crosstalks with the VEGF pathways. We will conduct in vivo experiments in mice to quantitate the role of TSP1, with and without administration of neutralizing agents for TSP1 and its receptors. The results will serve a basis for novel therapeutic approaches to PAD. Specific Aim 3 is devoted to characterization of the impact of pre- existing vascular rarefaction. Human PAD is a chronic disease that follows years of atherosclerotic buildup, resulting in vascular rarefaction in the affected distal muscle, whereas current mouse models of PAD do not have this pre-existing lower vascular density, thereby artificially increasing the perceived effectiveness of therapies. We will focus on later stages of the mouse PAD model, instead of the more common acute stage. The project should lead to important new knowledge and to novel therapeutic strategies in PAD.